Uncomplicated malaria Although long-lived classical MBCs (CD19+/CD20+/CD21+/CD27+/CD10-) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical memory B cells (MBCs) (CD19+/CD20+/CD21-/CD27-/CD10-). Similar expansions of atypical MBCs have been described for a variety of chronic infectious diseases including AIDS. At present, the function of atypical MBCs in malaria is not known nor are the factors that drive their differentiation. We showed that atypical MBCs have lost two key adaptive immune cell functions, namely the ability to signal through the BCR in response to soluble antigen and to differentiate into Ab secreting cells under a variety of conditions. To better understand the relationship between B cell subsets in malaria, we established a collaboration with Dr. Jun Zhu (NHLBI), an expert in RNAs as regulators of cellular differentiation, to carry out single cell RNA seq analysis of B cells from adults from malaria-endemic Mali. In a collaboration with Dr. Susan Moir (NIAID) we also compared our single cell RNAseq results to those of B cells from HIV high viremic individuals. From this analysis we made several important findings. We learned that: the atypical MBCs were composed of two different functional subpopulations defined by the isotype of their BCR; atypical and classical MBCs were derived from naive B cells; and that B cell subpopulations in malaria and HIV are remarkably similar. We completed a study to define the factors that drive the differentiation of human tonsil B cells to acquire the phenotype of atypical MBCs in vitro. We determined that predominantly naive B cells gave rise to T-bet + atypical MBCs. Key factors that triggered differentiation included: the form in which antigen was presented to B cells with presentation on membranes being most effective, and the presence of gamma IFN and the TLR9 agonist CpG. Provided with these stimuli naive B cells expressed a variety of genes associated with atypical MBCs including T-bet and FcRL5 and lost their ability to be triggered through the BCR. We showed earlier that atypical MBCs were unable to respond to soluble antigen. We asked are atypical MBCs able to respond to membrane associated antigens? We discovered that atypical MBCs are able to robustly signal in response to membrane-associated antigens and to capture and process the antigen. We determined that when engaging membrane-associated antigens B cells spread over the membrane and in the spreading process inhibitory receptors, that are highly expressed on atypical MBCs, were segregated away from the BCRs, allowing BCRs to function. We are pursuing this observation to determine the generality of the finding in different B cell subsets. We are testing this novel hypothesis using human Tfh cells and atypical MBCs in vitro. We also collaborated on malaria projects with NIAID investigators to determine the role of NK cells in inhibiting Pf growth in infected red blood cells by ADCC and the contribution of NK cells to the acquisition of protective immunity in children in malaria endemic Africa. Cerebral malaria In recent years we have used a mouse model of cerebral malaria (CM), termed experimental CM (ECM) to identify potential adjunctive therapies that could be used in African children. ECM exhibits many of the same features in CM including parasite infected red blood cells in the brain vasculature, brain swelling, hemorrhaging and breakdown of the blood brain barrier. In addition accumulation of CD8+ T cells in the brain has been shown to play a critical role in ECM disease. However, at present a role for CD8+ T cells in CM has not been rigorously investigated. We have tested a number of inhibitors of T cell metabolism as adjunctive therapy for CM in collaboration with Dr. Jonathan Powell (Johns Hopkins University). Remarkably we found that a glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), rescued mice from ECM, when administered late in the infection when mice already showed neurological signs of the disease. At the time of treatment mice were suffering blood-brain barrier dysfunction, brain swelling and hemorrhaging accompanied by accumulation of parasite-specific CD8+ effector T cells and infected red blood cells in the brain and perturbation of brain metabolism. Remarkably, DON-treatment restored blood-brain barrier integrity, reduced brain swelling, decreased the function of activated effector CD8+ T cells in the brain and returned brain metabolites to uninfected levels. Our goal over the last year was to better understand the similarities and differences in the pathology associated with CM in children in Malawi and ECM in mice to allow us to better evaluate DON as an adjunctive therapy in CM. To this end we established a collaboration with Dr. Terrie Taylor (Michigan State University), an expert in CM in children who heads a pediatric clinic in Malawi that treats children with CM. We described the immune infiltrates into the brains of children who died of CM using multiplex immunohistochemistry analysis of brain sections. We have now established for the first time that CD8+ T cells sequester along the abluminal face of blood vessels in the brains of children with CM. This finding opens up a new world of possible adjunctive therapies and validates the use of DON as CM therapy. We have also established a collaboration with Dr. Dima Hammoud (NIAID), an expert in the application of MRI and PET imaging to the study of brain infections to image brains of mice with CM. We used longitudinal MR imaging to visualize brain pathology in ECM and the impact of DON on disease progression in mice. For the first time, we demonstrated in vivo the reversal of disease markers in symptomatic infected mice following DON treatment, including resolution of edema and blood brain barrier disruption, findings usually associated with fatal outcome in children and adults with CM. Our results support the premise that DON is a potential adjunctive treatment that could rescue children and adults from fatal CM.